Functionalized encapsulated fluorescent nanocrystals

ABSTRACT

Provided are a functionalized, encapsulated fluorescent nanocrystal comprising a liposome having encapsulated therein one or more fluorescent nanocrystals; use of the functionalized, encapsulated fluorescent nanocrystals in detection systems; and a method of producing functionalized, encapsulated fluorescent nanocrystals. A method of using the functionalized encapsulated fluorescent nanocrystals having affinity molecule bound thereto comprises contacting the functionalized encapsulated fluorescent nanocrystals with a sample so that complexes are formed between the functionalized encapsulated fluorescent nanocrystals and substrate for which the affinity molecule has binding specificity, if the substrate is present; exposing the complexes in the detection system to an excitation light source, and detecting a fluorescence peak emitted from the complexes, if present.

This application is a continuation of U.S. application Ser. No.09/783,459 filed Feb. 12, 2001, now U.S. Pat. No. 6,761,877 which isincorporated herein by reference and further claims priority to theprovisional application Nos. 60/183,607 and 60/183,608 both filed onFeb. 18, 2000, all of which are herein incorporated by reference.

FIELD OF INVENTION

This invention relates generally to novel compositions comprisingencapsulated, fluorescent nanocrystals. More particularly, the presentinvention relates to the use of a vesicle or capsid to encapsulatefluorescent nanocrystals in forming water-soluble fluorescentnanocrystals.

BACKGROUND OF THE INVENTION

Nonisotopic detection systems have become a preferred mode in scientificresearch and clinical diagnostics for the detection of biomoleculesusing various assays including, but not limited to, flow cytometry,nucleic acid hybridization, DNA sequencing, nucleic acid amplification,microarrays, immunoassays, histochemistry, and functional assaysinvolving living cells. In particular, while fluorescent organicmolecules such as fluorescein and phycoerythrin are used frequently indetection systems, there are disadvantages in using these molecules incombination. For example, each type of fluorescent molecule typicallyrequires excitation with photons of a different wavelength as comparedto that required for another type of fluorescent molecule. However, evenwhen a single light source is used to provide a single excitationwavelength (in view of the spectral line width), often there isinsufficient spectral spacing between the emission optima of differentfluorescent molecules to permit individual and quantitative detectionwithout substantial spectral overlap.

Additionally, conventional fluorescent molecules have limitedfluorescence intensity. Further, currently available nonisotopicdetection systems typically are limited in sensitivity due to the finitenumber of nonisotopic molecules which can be used to label a biomoleculeto be detected.

Doped metal oxide (“DMO”) nanocrystals are nanocrystals that can beexcited with a single excitation light source resulting in a detectablefluorescence emission of high quantum yield (e. g., a single quantum dothaving at a fluorescence intensity that may be a log or more greaterthan that a molecule of a conventional fluorescent dye) and with adiscrete fluorescence peak. Typically, they have a substantially uniformsize of less than 200 Angstroms, and preferably have a substantiallyuniform size in the range of sizes of from about 1 nm to about 5 nm, orless than 1 nm.

In that regard, dMO nanocrystals are preferably comprised of metaloxides doped with one or more rare earth elements, wherein the dopantcomprising the rare earth element is capable of being excited (e. g.,with ultraviolet light) to produce a narrow spectrum of fluorescenceemission (typi-cally more narrow than the spectrum of fluorescenceemission emitted by a semiconductor nanocrystal). Such dMO nano-crystalsare well known in the art. However, a desirable feature of dMOnanocrystals when used for nonisotopic detection applications is thatthe nanocrystals be made water-soluble. “Water-soluble” is used hereinto mean that the nanocrystals are sufficiently soluble or suspendable inan aqueous-based solution including, but not limited to, water,water-based solutions, and buffer solutions, that are used in detectionprocesses, as known by those skilled in the diagnostic art.

Semiconductor nanocrystals are quantum dots that can be excited with asingle excitation light source resulting in a detectable fluorescenceemission of high quantum yield (e.g., a single quantum dot having at afluorescence intensity that may be a log or more greater than that amolecule of a conventional fluorescent dye) and with a discretefluorescence peak. Typically, they have a substantially uniform size ofless than 200 Angstroms, and preferably have a substantially uniformsize in the range of sizes of from about 1 nm to about 5 nm, or lessthan 1 nm. In that regard, quantum dots are preferably comprised of aGroup II-VI semiconductor material (of which ZnS, and CdSe areillustrative examples), or a Group III-V semiconductor material (ofwhich GaAs is an illustrative example). Such quantum dots are well knownin the art. However, a desirable feature of quantum dots when used fornonisotopic detection applications is that the quantum dots be madewater-soluble. Current methods of making semiconductor nanocrystalswater-soluble is to add to the semiconductor nanocrystal a layercomprising mercaptocarboxylic acid (Chen and Nie, 1998, Science281:2016–2018), or silica (U.S. Pat. No. 5,990,479), or one or morelayers of amino acids (U.S. Pat. No. 6,114,038). Depending on whichlayer composition is used, the treated nanocrystal may have limitedstability in an aqueous solution, particularly when exposed to air(oxygen) and/or light. More particularly, oxygen and light can cause themolecules comprising the layer to become oxidized, thereby formingdisulfides which destabilize the attachment of the layer molecules tothe semiconductor nanocrystals. Thus, oxidation may cause the layermolecules to become detached from the surface of the quantum dots,there-by exposing the surface of the quantum dots which may result in“destabilized quantum dots”. Destabilized quantum dots form aggregateswhen they interact together, and the formation of such aggregateseventually leads to irreversible flocculation of the quantum dots.Additionally, depending on the layer composition, it can causenon-specific binding, particularly to one or more molecules in a sampleother than the target molecule, which is not desirable in a detectionassay.

Hence, there is a need to provide alternative forms of water-soluble,fluorescent nanocrystals.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to provide fluorescentnanocrystals which are encapsulated by a vesicle or capsid comprising aliposome.

It is another object of the present invention to provide fluorescentnanocrystals which are encapsulated by or trapped within a vesicle orcapsid comprising a liposome, and wherein the surface of the liposome isfunctionalized with surface groups comprising a reactive functionalitythat may be used to form a bond with one or more molecules of anaffinity molecule which has a reactive functionality which is capable offorming a bond with the surface groups of the liposome.

It is another object of the present invention to provide a fluorescentnanocrystal which comprises one or more fluorescent nanocrystalsencapsulated by or trapped within a liposome, and wherein the surface ofthe liposome is functionalized with surface groups comprising one ormore reactive functionalities.

It is another object of the present invention to provide afunctionalized, encapsulated fluorescent nanocrystal which comprises oneor more fluorescent nanocrystals encapsulated by or trapped within aliposome which is functionalized by the addition of one or more affinitymolecules.

It is further object of the present invention to provide afunctionalized, encapsulated fluorescent nanocrystal which comprises oneor more fluorescent nanocrystals encapsulated by or trapped within aliposome, and wherein the liposome portion may be disrupted to releasethe fluorescent nanocrystals in a method of “quenching” the fluorescencein a reaction.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

By the term “fluorescent nanocrystals” is meant, for purposes of thespecification and claims to refer to fluorescent nanocrystals comprisedof doped metal oxide nanocrystals, semiconductor nanocrystals, or acombination thereof.

By the terms “doped metal oxide nanocrystals” or “dMO nanocrystals” ismeant, for purposes of the specification and claims to refer tonanocrystals comprised of: a metal oxide, and a dopant comprised of oneor more rare earth elements. For example, suitable metal oxides include,but are not limited to, yttrium oxide (Y₂O₃), zirconium oxide (ZrO₂),zinc oxide (ZnO), copper oxide (CuO or Cu₂O), gadolinium oxide (Gd₂O₃),praseodymium oxide (Pr₂O₃), lanthanum oxide (La₂O₃), and alloys thereof.The rare earth element comprises an element selected from the Lanthanideseries and includes, but is not limited to, europium (Eu), cerium (Ce),neodymium (Nd), samarium (Sm), terbium (Tb), gadolinium (Gd), holmium(Ho), thulium (Tm), an oxide thereof, and a combination thereof. Asknown to those skilled in the art, depending on the dopant, an energizeddMO nanocrystal is capable of emitting light of a particular color.Thus, the nature of the rare earth or rare earths are selected inconsequence to the color sought to be imparted (emitted) by afunctionalized, encapsulated dMO nanocrystal according to the presentinvention. A given rare earth or rare earth combination has a givencolor, thereby permitting the provision of functionalized, encapsulateddMO nanocrystals, each of which may emit (with a narrow emission peak) acolor over an entire range of colors by adjusting the nature of thedopant, the concentration of the dopant, or a combination thereof. Forexample, the emission color and brightness (e.g., intensity) of a dMOnanocrystal comprising Y₂O₃:Eu may depend on the concentration of Eu;e.g., emission color may shift from yellow to red with increasing Euconcentration. For purposes to illustration only, representative colorswhich may be provided are listed in Table 1.

TABLE 1 Fluorescent Color Dopant Blue thulium Blue cerium yellow-greenterbium Green holmium Green erbium Red europium reddish orange samariumOrange neodymium Yellow dysprosium White praseodymium orange-yelloweuropium + terbium orange-red europium + samariumMethods for making dMO nanocrystals are known to include, but are notlimited to a sol-gel process (see, e.g., U.S. Pat. No. 5,637,258), andan organometallic reaction. As will be apparent to one skilled in theart, the dopant (e.g., one or more rare earth elements) are incorporatedinto the dMO nanocrystal in a sufficient amount to permit the dMOnanocrystal to be put to practical use in fluorescence_detection asdescribed herein in more detail. An insufficient amount comprises eithertoo little dopant which would fail to emit sufficient detectablefluorescence, or too much dopant which would cause reduced fluorescencedue to concentration quenching. In a preferred embodiment, the amount ofdopant in a dMO nanocrystal is a molar amount in the dMO nanocrystalselected in the range of from about 0.1% to about 25%.

By the term “semiconductor nanocrystals” is meant, for purposes of thespecification and claims to refer to quantum dots (crystallinesemiconductors) comprised of a core comprised of at least one of a GroupII-VI semiconductor material (of which ZnS, and CdSe are illustrativeexamples), or a Group III-V semiconductor material (of which GaAs is anillustrative example), a Group IV semiconductor material, or acombination thereof. In a preferred embodiment, the core of the quantumdots may be passivated with an semiconductor overlayering (“shell”)uniformly deposited thereon. For example, a Group II-VI semiconductorcore may be passivated with a Group II-VI semiconductor shell (e.g., aZnS or CdSe core may be passivated with a shell comprised of YZ whereinY is Cd or Zn, and Z is S, or Se). As known to those skilled in the art,the size of the semiconductor core correlates with the spectral range ofemission, as illustrated in Table 1 for CdSe.

TABLE 1 Color Size Range (nm) Peak Emission Range blue  2.5 to 2.68 476to 486 green 2.8 to 3.4 500 to 530 yellow 3.58 to 4.26 536 to 564 orange4.9 to 6.1 590 to 620 red  8.6 to 10.2 644 to 654Methods for making semiconductor nanocrystals are known in the art. Apreferred method of making semiconductor nanocrystals is by a continuousflow process (U.S. Pat. No. 6,179,912, the disclosure of which is hereinincorporated by reference).

By the term “affinity molecule” is meant, for purposes of thespecification and claims, to mean a molecule which is capable of bindingto another molecule; and in a preferred embodiment, has bindingspecificity and avidity for a target molecule. In general, affinitymolecules are known to those skilled in the art to include, but are notlimited to, lectins or fragments (or derivatives) thereof which retainbinding function; monoclonal antibodies (“mAb”, including chimeric orgenetically modified monoclonal antibodies (e.g., “humanized”));peptides; aptamers; nucleobases (synthetic, natural, or modified);nucleic acid molecules (including, but not limited to, single strandedRNA or single-stranded DNA, or single-stranded nucleic acid hybrids);avidin, or streptavidin, or avidin derivatives; and the like. Theinvention may be practiced using a preferred affinity molecule to theexclusion of affinity molecules other than the preferred affinitymolecule. The term “monoclonal antibody” is also used herein, forpurposes of the specification and claims, to include immunoreactivefragments or derivatives derived from a mAb molecule, which fragments orderivatives retain all or a portion of the binding function of the wholemAb molecule. Such immunoreactive fragments or derivatives are known tothose skilled in the art to include F(ab′)₂, Fab′, Fab, Fv, scFV, Fd′and Fd fragments. Methods for producing the various fragments orderivatives from mAbs are well known in the art. The construction ofchimeric antibodies is now a straightforward procedure in which thechimeric antibody is made by joining the murine variable region to ahuman constant region. Additionally, “humanized” antibodies may be madeby joining the hypervariable regions of the murine monoclonal antibodyto a constant region and portions of variable region (light chain andheavy chain) sequences of human immunoglobulins using one of severaltechniques known in the art. Aptamers can be made using methodsdescribed in U.S. Pat. No. 5,789,157 (herein incorporated by reference).Lectins, and fragments thereof, are commercially available. Lectins areknown to those skilled in the art, and are commercially available.

By the term “nucleobase” is meant, for purposes of the specification andclaims to refer to a nucleic acid moiety including, but not limited to:nucleosides (including derivatives, or functional equivalents thereof,and synthetic or modified nucleosides, and particularly, a nucleosidecomprising a reactive functionality (e.g., free amino group or carboxylgroup); nucleotides (including dNTPs, ddNTPs, derivatives or functionalequivalents thereof, and particularly, a nucleotide comprising areactive functionality (e.g., free amino group or carboxyl group);acyclonucleoside triphosphates (see, e.g., U.S. Pat. No. 5,558,991);3′(2′)-amino-modified nucleosides, 3′(2′)-amino-modified nucleotides,3′(2′)-thiol-modified nucleosides, 3′(2′)-thiol-modified nucleotides(see, e.g., U.S. Pat. No. 5,679,785); alkynylamino-nucleotides (see,e.g., as a chain terminator, U.S. Pat. No. 5,151,507); and nucleosidethiotriphosphates (see, e.g., U.S. Pat. No. 5,187,085).

By the term “reactive functionality” is meant, for purposes of thespecification and claims, to refer to a free chemical group which canbond or associate with a chemical-reactive group (reactive with the freechemical groups). In a preferred embodiment, the resultant bond orassociation is of sufficient stability to withstand conditionsencountered in a method of detection, as known in the art. Free chemicalgroups include, but are not limited to a thiol, carboxyl, hydroxyl,amino, amine, sulfo, phosphate, or the like; whereas chemical-reactivegroups include, but are not limited to, thiol-reactive group,carboxyl-reactive group, hydroxyl-reactive group, amino-reactive group,amine-reactive group, sulfo-reactive group, or the like.

By the term “liposome” is meant, for purposes of the specification andclaims, to refer to a generally spherical vesicle or capsid generallycomprised of amphipathic molecules (e.g., having both a hydrophobic(nonpolar) portion and a hydrophilic (polar) portion). Typically, theliposome can be produced as a single (unilamellar) closed bilayer or amulticompartment (multilamellar) closed bilayer. The liposome can beformed by natural lipids, synthetic lipids, or a combination thereof. Ina preferred embodiment, the liposome comprises one or morephospholipids. In a more preferred embodiment, the liposome issubstituted with one or more conventional additives (“a component forsubstitution”), wherein the one or more additives are selected from thegroup consisting of a membrane stabilizer, an isotonic agent (e.g.,sugars, sodium chloride, polyalcohols such as mannitol, sorbitol, andthe like), a pH adjusting agent (e.g., a base, a basic amino acid, anacidic amino acid, sodium phosphate, sodium carbonate, and the like,present in an amount to adjust the liposome to a desired pH), anaggregation minimizer (e.g., a surfactant (e.g., polysorbates,poloxamers), polysaccharide, liposomal surface carboxyl groups, and thelike), an affinity molecule, an amino acid, and a combination thereof.As apparent to one skilled in the art, and depending on the lipidcomposition and the composition of the component for substitution, theone or more components for substitution may be added during theformation of the liposome, may be added after the formation of theliposome, or a combination thereof. A preferred component forsubstitution of the liposome may be used to the exclusion of componentsother than the preferred component. For example, a membrane stabilizeris added in an effective amount to increase the stability of a liposome.Stability refers to one or more of membrane integrity, ability towithstand heat (e.g., a temperature above room temperature, andpreferably a temperature in the range of from about 35° C. to about 100°C.), ability to withstand oxygen (e.g., as exposed during normal useconditions), ability to withstand light (e.g., as exposed during normaluse conditions), and a combination thereof. A membrane stabilizer maycomprise one or more sterols (e.g., cholesterol), one or more fattyacids, one or more amino acids, and a combination thereof. Also,stability, with respect to exposure to oxidation, may be enhanced bynitrogen gas substitution using methods known on the art. Lipids knownin the art for forming liposomes include, but are not limited to,lecithin (soy or egg; phosphatidylcholine),dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine,distearoylphosphatidylcholine, dicetylphosphate, phosphatidylglycerol,hydrogenated phosphatidylcholine, phosphatidic acid, cholesterol,phosphatidylinositol, a glycolipid, phosphatidylethanolamine,phosphatidylserine, a maleimidyl-derivatized phospholipid (e.g.,N-[4(p-malei-midophenyl)butyryl]phosphatidylethanolamine),dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol,dimyristoylphosphatidic acid, and a combination thereof. It will beapparent to one skilled in the art that the ratio of the one or morelipids and the one or more components for substitution will depend onfactors including, but not limited to, the composition of the lipids,the intended function of each lipid (e.g., the reason for its inclusionin the liposome), the composition of the component for substitution, theintended function of each component for substitution (e.g., the reasonfor its inclusion in the liposome), and the desired properties of theliposome portion of a functionalized, encapsulated fluorescentnanocrystal (e.g., size of interior space or “capture volume”, pH rangeof stability, temperature range of stability, desired surface charge,desired surface free chemical group). In that regard, as known to thoseskilled in the art, a particular lipid or lipid combination, when usedto form a liposome, can offer particular benefits. For example,inclusion of phosphatidylglycerol in combination with other lipids(e.g., with phosphatidylcholine and cholesterol, ratio of 1:9:8) impartsa negative charge to the liposome which increases intralamellar spacing(capture volume), reduces aggregation, and facilitates initial hydrationof the lipid. In a preferred embodiment, the liposomes encapsulating thefluorescent nanocrystals are stable at a neutral pH of from about 6 toabout 7; and in a more preferred embodiment, are stable in a broad pHrange of from about 4 to about 12. A preferred liposome (content andcomposition) may be formed as part of the functionalized, encapsulatedfluorescent nanocrystals according to the present invention to theexclusion of liposomes other than the preferred liposome.

By the term “functionalized, encapsulated fluorescent nanocrystal” ismeant, for purposes of the specification and claims to refer to one ormore fluorescent nanocrystals which have been encapsulated (e.g.,without establishing a chemical linkage or bond between the one or morefluorescent nanocrystals and the liposome) by a liposome; wherein theouter surface of the liposome is functionalized with surface groupscomprising one or more reactive functionalities, one or more affinitymolecules, or a combination thereof; and wherein the functionalized,encapsulated fluorescent nanocrystal is water-soluble. In one preferredembodiment, a single fluorescent nanocrystal is encapsulated by theliposome. In another preferred embodiment, a plurality of fluorescentnanocrystals are encapsulated by a liposome. It will be apparent to oneskilled in the art that the number of fluorescent nanocrystalsencapsulated per liposome can be controlled by factors that include, butare not limited to, the size of the liposome formed, the method in whichthe fluorescent nanocrystals are encapsulated, post productionprocessing by size exclusion, and the ratio of fluorescent nanocrystalsto lipid mixture during formation. It will also be apparent to oneskilled in the art, that where a plurality of fluorescent nanocrystalsare encapsulated by a liposome, the fluorescent nanocrystals may behomogeneous (i.e., capable of fluorescing essentially the same color) ormay be heterogenous (e.g., comprising different populations wherein eachpopulation is capable of fluorescing a different (spectrallydistinguishable) color than another population of fluorescentnanocrystal that is encapsulated).

By the term “strand synthesis” is meant for purposes of thespecification and claims to refer to the production of one more strands,or portions thereof, such as through enzymatic copying by an enzymewhich replicates nucleic acids in a template-directed manner. There isno particular size, length or content limitations for the strand. Thus,“strand synthesis” encompasses processes including, but not limited to,nucleic acid amplification, DNA sequencing, fill-in reactions, reversetranscription, in vitro mutagenesis, cycled chain termination sequencereactions, cycled primer extension reactions, random primer extensionreactions, nick translations, primer elongation, methods for determiningthe presence and quantifying the number of di- and trinucleotide repeats(see, e.g., U.S. Pat. No. 5,650,277), and DNA typing with short tandemrepeat polymorphisms (see, e.g., U.S. Pat. No. 5,364,759). The nucleicacid composition of the strand synthesized may be selected frommolecules which include nucleobases; and more preferably,ribonucleotides (RNA), or deoxyribonucleotides (DNA).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compositions comprising functionalized,encapsulated fluorescent nanocrystals. Preferably, the functionalized,encapsulated fluorescent nanocrystals are water-soluble, and can bestored without significant leakage (of the one or more fluorescentnanocrystals from the liposome) over long periods of time. The outersurface of the liposome portion (e.g., polar head groups in contact withthe surrounding aqueous environment) is functionalized with surfacegroups comprising one or more reactive functionalities, one or moreaffinity molecules, or a combination thereof. As known to those skilledin the art, dMO nanocrystals and semiconductor nanocrystals aregenerally soluble in organic solvents, and have limited or no solubilityin aqueous environments. Thus, a method for removing fluorescence fromthe aqueous environment containing functionalized, encapsulatedfluorescent nanocrystals comprises contacting the functionalized,encapsulated fluorescent nanocrystals with an effective amount of adisrupting agent (“lipolytic agent”) to disrupt the liposome portion ofthe functionalized, encapsulated fluorescent nanocrystals therebyreleasing the fluorescent nanocrystals into the aqueous environment withresultant precipitation out of solution.

As general guidance for producing functionalized, encapsulatedfluorescent nanocrystals according to the present invention, there arevarious methods for forming liposomes which may be suitable forencapsulating fluorescent nanocrystals. In one embodiment, the lipids(e.g., phospholipids and sterol) for forming the liposomes, and thefluorescent nanocrystals to be encapsulated, are dissolved in a suitablesolvent (e.g., chloroform), and the solvent is evaporated in vacuo toresult in a film comprising the lipids and fluorescent nanocrystals(“dried lipid mixture film”). For example, the lipids, fluorescentnanocrystals, and the organic solvent may be added to and mixed in arotoevaporator flask, and dried under vacuum in a rotary evaporatoruntil the contents form a thin homogenous film. Alternatively, a driedlipid mixture film may be formed by forming a dried lipid film, andadding to that dried lipid film a dried preparation of fluorescentnanocrystals. An aqueous solution is then added to the dried lipidmixture film, and the film is allowed to hydrate (e.g., for between10–30 minutes at room temperature) resulting in spontaneous formation ofliposomes which encapsulate fluorescent nanocrystals. The lipiddispersion may then be vigorously vortexed (e.g., 45 to 60 minutes) tofacilitate continued formation of functionalized, encapsulatedfluorescent nanocrystals. If desired, the lipid bilayers may be annealedby heating the dispersion to about 45 to 50° C. followed by a gradualcooling to about 4° C. It will be apparent to one skilled in the artthat one or more components for substitution of the liposome may besuspended in the aqueous solution prior to the addition of the aqueoussolution to the dried lipid mixture film. Alternatively, the liposomeportion of the functionalized, encapsulated fluorescent nanocrystals maybe post-treated (treated subsequent to formation) with one or morecomponents for substitution of the liposome portion. For example, anaqueous solution containing the one or more components for substitutionmay be in prolonged contact (e.g., incubated overnight) with thefunctionalized, encapsulated fluorescent nanocrystals. As anotheralternative embodiment, the aqueous solution containing the one or morecomponents for substitution, and dried lipid mixture film may be mixedtogether and strongly vortexed, followed by extrusion of the mixtureunder pressure through a membrane filter (e.g., polycarbonate) of adesired pore size to obtain a solution containing functionalized,encapsulated fluorescent nanocrystals. In another alternativeembodiment, the one or more components for substitution are suspended inan aqueous solution, and then lyophilized. The lyophilized residue isthen dissolved in a glycerol buffer (e.g., a 2% glycerol solutioncontaining 0.5 mM EDTA, pH 6.0), and filtered through a membrane filter(e.g., polycarbonate) of a desired pore size. The resultant filtrate isadded to the dried lipid mixture film, and the resultant mixture is thenhydrated with an aqueous solution and vortexed to form functionalized,encapsulated fluorescent nanocrystals. If desired, the formedfunctionalized, encapsulated fluorescent nanocrystals may then beextruded through a membrane filter (e.g., polycarbonate) of a desiredpore size. In any of these embodiments, the functionalized, encapsulatedfluorescent nanocrystals would remain soluble in the aqueous solution inwhich they are formed, whereas unencapsulated fluorescent nanocrystalsmay eventually precipitate; hence, a purification may be achievedbetween functionalized, encapsulated fluorescent nanocrystals andunencapsulated fluorescent nanocrystals.

As will be apparent to one skilled in the art, there are various knownmethods for producing liposomes that may also be useful for producingfunctionalized, encapsulated fluorescent nanocrystals. Such methods mayinclude, but are not limited to, a vortexing method, an ultrasonicmethod, an extrusion method, a reverse-phase evaporation method, asolvent injection method, a surfactant (e.g., detergent)-removal method,an annealing method, and a forced extrusion following freeze-thawcycles. Each method may offer an advantage; thus, a combination ofmethods may be desirable (for a review, see, Szoka and Papahadjopoulos,1981, Chapter 3 in “Liposomes: From Physical Structure to TherapeuticApplications”, the contents of which are herein incorporated byreference). For example, sonication is a method used to produceliposomes of a relatively small size as compared to other methods;however, the size is largely heterogenous. By extrusion through amembrane of a defined size, or series of membranes with pores ofdecreasing diameter, size heterogeneity can be reduced, therebyresulting in liposomes of a well-defined and narrow size dispersion. Inanother example, sonication may result in liposomes incorporatingstructural defects. However, annealing (at a temperature above the T_(c)of the highest melting lipid in the mixture used to form the liposome;e.g., for 30 minutes) can stabilize liposomes (note though, annealing isgenerally not effective when the liposome is composed of an equimolarratio of phospholipid and cholesterol). The above principles can beapplied to methods for producing functionalized, encapsulatedfluorescent nanocrystals.

The selection and molar ratio of the combination of lipids, with orwithout one or more components for substitution, for encapsulatingfluorescent nanocrystals may depend on factors which include, but arenot limited to, the application in which the functionalized,encapsulated fluorescent nanocrystals are to be used, the desiredsurface groups comprising one or more reactive functionalities, desiredsize and/or stability and/or surface charge of the functionalized,encapsulated fluorescent nanocrystals, and the one or more methods usedto make the functionalized, encapsulated fluorescent nanocrystals.Although various combinations may be used, a preferred combination maybe selected from two general groupings of suitable lipid mixtures forforming the functionalized, encapsulated fluorescent nanocrystalsaccording to the present invention: a combination of phospholipids witha sterol, wherein a phospholipid in the greatest amount of thecombination (as compared to the amounts of the one or more remainingphospholipids of the combination) is in approximate equimolar ratio withthe sterol; and a combination of phospholipids with a sterol, whereinthe sterol is not in approximate equimolar ratio with the phospholipidcomprising the highest amount (concentration) in the combination (ascompared to the amounts of the one or more remaining phospholipids ofthe combination). Either combination may further comprise one or morecomponents for substitution of the liposome portion of thefunctionalized, encapsulated fluorescent nanocrystals, as describedherein in more detail. For purposes of illustration only, and notlimitation, combinations of lipids (including with exemplary molarratios) that may be useful in making the compositions according to thepresent invention include, but are not limited to, phosphatidylcholine(“PC”)/cholesterol (“ch”)/phosphatidylserine (“PS”), 5:4:1;PC/ch/phosphatidylglycerol (“PG”), 8:2:1.2 or 9:8:1 or 9:5:1;PC/ch/phosphatidylethanolamine, 6:2:2 or 5:4:1; anddipalmitoylPC/ch/phosphatidic acid, 7:2:1. In a preferred embodiment, acombination of lipids may further comprise one or more components forsubstitution which is added to the lipids in parts by weight (asexpressed in relation to the lipid mixture wherein the total lipidmixture comprises 1 part by weight) in a range of from about 0.0001 toabout 0.5, depending on the nature of the one or more components, andthe intended function. A preferred combination of lipids and one or morecomponents for substitution may be used to the exclusion of combinationsother than the preferred combination in producing the functionalized,encapsulated fluorescent nanocrystals according to the presentinvention. Similarly, a preferred fluorescent nanocrystal may be used tothe exclusion of a fluorescent nanocrystal other than the preferredfluorescent nanocrystal in producing the functionalized, encapsulatedfluorescent nanocrystals according to the present invention.

In another preferred embodiment, the one or more affinity moleculesdesired to be incorporated as part of a functionalized, encapsulatedfluorescent nanocrystals is added in the process of producingfunctionalized, encapsulated fluorescent nanocrystals. In this preferredembodiment, it is desirable that the affinity molecule be comprised of ahydrophobic portion and a hydrophilic portion so that its hydrophobicportion will facilitate interaction with the hydrophobic portion of thelipids in the lipid mixture in forming the liposome portion of thefunctionalized, encapsulated fluorescent nanocrystals; and itshydrophilic portion will extend out from the surface of thefunctionalized, encapsulated fluorescent nanocrystals. For example, adried lipid mixture comprising dried lipids, a dried preparation of thefluorescent nanocrystals, and a dried (e.g., lyophilized) preparation ofthe affinity molecule comprising a protein (e.g., monoclonal antibody,or peptide, or glycoprotein, or lipoprotein, etc.) is hydrated by theaddition of an aqueous solution (alternatively, the affinity molecule issuspended in the aqueous solution); and the resultant dispersion is thenvigorously vortexed to facilitate formation of functionalized,encapsulated fluorescent nanocrystals which have incorporated in theliposome portion the one or more affinity molecules. It will be apparentto one skilled in the art that, as compared to neutral phospholipids(e.g., PC), anionic phospholipids (e.g., PG and PS) enhance the bindingof the affinity molecule in the liposome portion of the functionalized,encapsulated fluorescent nanocrystals. It will be apparent to oneskilled in the art that the amount of affinity molecule to beincorporated, and the content (ratio) and composition of the lipidmixture will depend on the specific affinity molecule to be incorporatedas well as the desired application of use for the functionalized,encapsulated fluorescent nanocrystals. In an illustrative, non-limitingexample, the lipid mixture may comprise PC/ch/PG (17:5:2.5) and theaffinity molecule comprises a peptide in an amount that is in a range offrom about 0.001 mg/ml to about 1 mg/ml. As described in more detailherein, one or more components for substitution may be added.Additionally, if desired the functionalized, encapsulated fluorescentnanocrystals may be subjected to a purification process such as sizeexclusion, or separation by function, or other method known in the artfor purification.

The following examples are provided to further describe the invention,but are not to be considered limitative of the invention.

EXAMPLE 1

This example is a non-limiting illustration of a method of making thefunctionalized, encapsulated fluorescent nanocrystals according to thepresent invention. dMO nanocrystals comprising yttrium oxide doped witheuropium were encapsulated to form functionalized, encapsulated dMOnanocrystals. Briefly, this was performed as follows. A first solutionwas comprised of droplets of a solution comprising a metal salt (yttriumsalt and europium salt) solubilized in water, a surfactant (non-ionic,cationic or ionic) and an oil (e.g., octane). The surfactant can be alipid mixture or other suitable bipolar molecule (e.g.,cetyltrimethylammonium bromide). The approximate ratio of the metal saltsolution:surfactant:oil was 10:15:85. A second solution was comprised ofdroplets of a solution comprising a hydroxide (ammonium hydroxide)solubilized in water, a surfactant, and an oil, at an approximate ratioof 10:15:85. The first solution and the second solution were mixed(e.g., stirred), thereby resulting in the formation of fluorescentnanocrystals comprised of yttrium oxide doped with europium, and theencapsulation of the fluorescent nanocrystals. The ratio of metal saltin the first solution to hydroxide in the second solution wasapproximately 1:2. The mixed solution was the centrifuged to pellet thefunctionalized, encapsulated fluorescent nanocrystals. Thefunctionalized, encapsulated fluorescent nanocrystals were then washedto remove loose surfactant and oil (followed by pelleting), and thenresuspended in water.

If desired, the suspension comprising functionalized, encapsulatedfluorescent nanocrystals may be further purified (e.g., separatingfunctionalized, encapsulated fluorescent nanocrystals from liposomes notcontaining fluorescent nanocrystals) or selected for size using methodsknown in the art. For example, the suspension may be overlayed onto adensity gradient solution (e.g., sucrose, or glycerol, or Ficoll) andthen centrifuged for a sufficient time to achieve the desired separationof liposome species that may be present in the suspension. In continuingthis example, functionalized, encapsulated fluorescent nanocrystalswould have a greater density than liposomes not containing fluorescentnanocrystals. Hence, the density gradient may be exposed to anexcitation wavelength spectra, and the fluorescing band comprisingfunctionalized, encapsulated fluorescent nanocrystals may then beharvested from the rest of the density gradient. Alternatively,functionalized, encapsulated fluorescent nanocrystals may be furtherpurified using size exclusion chromatography, or by magnetic separation.

EXAMPLE 2

This example is a non-limiting illustration of a method of making thefunctionalized, encapsulated fluorescent nanocrystals according to thepresent invention. Semiconductor nanocrystals comprising CdSe core, ZnSshell, of a size of about 7.6 nm (core size of about 5.1 nm), and havinga peak emission spectra of about 606 nm, were prepared forencapsulation. The semiconductor nanocrystals (100 ul of a 7×10⁻⁶ Mpyridine solution) were precipitated in a microfuge tube with 500 ul ofhexanes. The mixture was centrifuged to pellet the semiconductornanocrystals, the hexanes/pyridine supernatant was discarded, and thepellet was then resuspended in 100 ul of DMSO (dimethyl sulfoxide). TheDMSO solution containing the semiconductor nanocrystals was mixed with0.9 ml of chloroform. The solution was then extracted twice with 500 ulof water to remove the DMSO. The solution was then centrifuged to removeinsoluble material, and the supernatant containing solubilizedsemiconductor nanocrystals was then decanted, and stored (4° C.) untiluse. To encapsulate the semiconductor nanocrystals, utilized was a lipidmixture comprising phosphatidylchloline (PC) and cholesterol (ch) at aratio of 5:2. Into a flask containing 1 ml of chloroform was added 100μl of the solution comprising the solubilized semiconductornanocrystals. With stirring, PC (15 mg) was added to the mixture, andthen ch (6 mg) was added-to the mixture. A schlenk adapter was attachedto the flask and the solvent contained therein was removed under vacuumto produce a dried lipid mixture film with semiconductor nanocrystals.To the film was added 3 ml of distilled water, and the solution was thensonicated in a water bath for 20 minutes. The resultant suspensioncontaining functionalized, encapsulated fluorescent nanocrystals wasextruded through a 0.2 um syringe filter. Examination by fluorescencemicroscopy confirmed formation of functionalized, encapsulatedfluorescent nanocrystals of a narrow size distribution.

If desired, the suspension comprising functionalized, encapsulatedfluorescent nanocrystals may be further purified (e.g., separatingfunctionalized, encapsulated fluorescent nanocrystals from liposomes notcontaining fluorescent nanocrystals) or selected for size using methodsknown in the art, as described in Example 1 herein in more detail. Itwill be apparent to one skilled in the art from the descriptions hereinthat a functionalized, encapsulated fluorescent nanocrystal may comprisea combination of one or more dMO nanocrystals and one or moresemiconductor nanocrystals (preferably, each population of nanocrystalbeing spectrally distinguishable from other populations of nanocrystalsin the combination of nanocrystals). Thus, for example, an organicsolution comprising dMO nanocrystals (e.g., comprising yttrium oxidedoped with europium and having a peak emission spectra that isspectrally distinguishable from the CdSe/ZnS nanocrystals having a peakemission spectra of about 606 nm) and semiconductor nanocrystals (e.g.,CdSe/ZnS nanocrystals having a peak emission spectra of about 606 nm)may be mixed with the phospholipids as described herein in producing adried lipid mixture film. The dried lipid film may then be treated, asdescribed in this Example 2, to produce functionalized, encapsulatedfluorescent nanocrystals.

EXAMPLE 3

This example illustrates functionalized, encapsulated fluorescentnanocrystals according to the present invention. As described herein inmore detail, encapsulated fluorescent nanocrystals may be functionalizedby comprising surface groups comprising one or more reactivefunctionalities which are capable of bonding to a reactive functionalityof, and which may be used to bond the liposome portion to, an affinitymolecule. Thus, a combination of reactive functionalities may be used tolabel an affinity molecule with a functionalized, encapsulatedfluorescent nanocrystal. If desired, as a separate step in the reaction,free reactive functionalities that may be present after the labelingprocess may be blocked or deactivated using methods known in the art(e.g., by adding a molecule which binds to the free reactivefunctionality). After the reaction in which the reactive functionalitycombination is used to perform the labeling, an affinity moleculelabeled with a functionalized, encapsulated fluorescent nanocrystal maybe separated from unbound affinity molecule and unbound (not bond toaffinity molecule) functionalized, encapsulated fluorescent nanocrystalby one or more methods known in the art including, but not limited tochromatography, and size fractionation. A preferred reactivefunctionality combination may be used to the exclusion of a reactivefunctionality combination other than the preferred reactivefunctionality combination.

3.1 Reactive Functionality Combination Comprising Amino Groups andAmino-reactive Groups.

In one preferred embodiment, the functionalized, encapsulatedfluorescent nanocrystals comprise surface groups comprising free aminogroups provided by a lipid, a component for substitution, or acombination thereof. In one preferred embodiment, the free amino groupsare provided by including an aminolipid in the lipid mixture which formsthe liposome portion of the functionalized, encapsulated fluorescentnanocrystals. For example, molecules of phosphatidylserine,phosphatidylethanolamine (“PE”), or dioleoyl PE, when incorporated inthe liposome portion, provides primary amino groups which are free toreact and chemically bond with one or more affinity molecules having oneor more amino-reactive groups. In one preferred embodiment, an affinitymolecule comprising a nucleobase having a reactive functionalitycomprising a free amino-reactive group (e.g., a carboxyl group) can bechemically bonded to a surface group comprising a free amino group usingmethods known in the art. Briefly, the nucleobase is esterified bytreatment with EDC (1-ethyl-3-[3-dimethyl-aminopropyl]carbdiimide)followed by treatment with sulfo-NHS at ambient temperature in bufferedaqueous solution (at about pH 7.4; e.g., for 30 minutes).2-mercaptoethanol is added to the solution (e.g., at a concentration of20 mM), and the mixture is stirred (e.g., for 15 minutes) to quench anyunreacted EDC. The activated nucleobase is then contacted with a molconcentration of the functionalized, encapsulated fluorescentnanocrystals (depending on the size, and desired number) for coupling anappropriate and desired number of nucleobase molecules (e.g., one, or ifdesired, more than one) to a functionalized, encapsulated fluorescentnanocrystal, and the reaction mixture is stirred (e.g., for 2 hours orreacted in other suitable conditions for forming an amide bond betweenthe EDC-activated carboxylates of the nucleobase molecules and the aminegroups of the functionalized, encapsulated fluorescent nanocrystals).Ethanolamine is then added (e.g., at a concentration of 30 mM) to quenchthe coupling reaction (e.g., with stirring for 30 minutes). Theresulting solution is then filtered and/or dialyzed to remove excessreagents. The result is the production of functionalized, encapsulatedfluorescent nanocrystals having a nucleobase covalently coupled thereto(e.g., a nucleobase labeled with a functionalized, encapsulatedfluorescent nanocrystal).

In another preferred embodiment, covalently coupled to surface groupscomprising the free amino groups of functionalized, encapsulatedfluorescent nanocrystals are nucleobases having free amino groups, viathe usage of a component for substitution comprising a spacer arm whichterminates at each end with an amino-reactive group. Spacer arms areknown in the art to include a chemical (glutaraldehyde), a hydrocarbonchain; and a peptide. For example, the surface groups comprising thefree amino groups may be activated by incubating the functionalized,encapsulated fluorescent nanocrystals with a solution containingglutaraldehyde (e.g., 25%, at 20° C. for 10 minutes), followed bydialysis to remove excess glutaraldehyde. The activated functionalized,encapsulated fluorescent nanocrystals are then contacted with a solutionof the nucleobase containing the free amino-reactive groups in a molconcentration for coupling the desired number of nucleobase molecules toa functionalized, encapsulated fluorescent nanocrystal. If desired, anyfree reactive functionalities may be blocked (e.g., by addition ofethanolamine). The resulting solution is then filtered and/or dialyzedto remove excess reagents. The result is the production offunctionalized, encapsulated fluorescent nanocrystals having anucleobase covalently coupled thereto (e.g., a nucleobase labeled with afunctionalized, encapsulated fluorescent nanocrystal).

3.2 Reactive Functionality Combination Comprising Thiol Groups andThiol-reactive Groups.

In another preferred embodiment, the nucleobase is labeled with (bondedor coupled to) a functionalized, encapsulated fluorescent nanocrystalaccording to the present invention by using reactive functionalitiescomprising thiol group and thiol-reactive groups.

3.2(a) For example, as previously described herein, the functionalized,encapsulated fluorescent nanocrystals may have surface groups comprisingfree amino groups. These surface groups may be further functionalized bythe addition (either in the presence or absence of EDC) of a maleimidederivative that reacts with amino groups. Such a maleimide derivativemay include, but is not limited to 3-maleimido-propionic acidN-hydroxysuccinimide ester, 3-maleimido-propionic acid,3-maleimidobenzoic acid N-hydroxysuccinimide ester,4-(maleimido-methyl)-1-cyclo-hexanecarboxylic acid N-hydroxysuccinimideester. The resultant functionalized, encapsulated fluorescentnanocrystals, having a thiol-reactive group, can interact with and bondto a nucleobase previously derivatized with one or more thiol groups.3.2 (b) As an alternative, the functionalized, encapsulated fluorescentnanocrystals having surface groups comprising free amino groups andmolecules of the nucleobase having thiol groups are mixed together inthe presence of a component for substitution comprising a cross-linkingreagent. The cross-linking reagent has an amino-reactive group at oneend, and a thiol-reactive group at the other end. Such cross-linkingreagents are known to those skilled in the art to include, but are notlimited to, sulfosuccinimidyl 6-[3′-(2pyridyldithio)-propionamido]hexonate,N-succini-midyl-[4-vinylsulfonyl]benzoate,sulfosuccinimidyl-[4-iodoacetyl]aminobenzoate,N-succinimidyl-[4-iodoacetyl]aminobenzoate, and N-succinimidyliodoacetate, succinimidyl 3-[bromoacetamido]propionate.

In either of the embodiments illustrated in 3.2(a) or (b), thefunctionalized, encapsulated fluorescent nanocrystals having reactivefunctionalities comprising a thiol-reactive group (e.g., as part of alipid or as a result of a cross-linking reagent) can be contacted with,in suitable conditions for bonding to, a nucleobase previouslyderivatized with one or more thiol groups. The suitable conditionsdepends on factors including whether a cross-linking reagent is used,the nature of the cross-linking reagent, and the desired number ofmolecules of functionalized, encapsulated fluorescent nanocrystals to belabeled to the affinity molecule. When using a commercial cross-linkingreagent, suitable conditions are often specified by the manufacturer.Nucleobases can be derivatized to include thiol groups using methodsknown to those skilled in the art (see, e.g., U.S. Pat. No. 5,679,785,herein incorporated by reference). For example, the OH group located inthe 3′ and/or 2′ position of a nucleobase may be derivatized to a thiolgroup, thereby allowing interaction with the thiol-reactive group of afunctionalized, encapsulated fluorescent nanocrystal, in formingthioether bonds that couple the nucleobase to the functionalized,encapsulated fluorescent nanocrystal. In another example, the3′-O-position and/or the 2′-O-position of a nucleobase may bederivatized to include alkylthiol chemical functionality, which then canbe treated with acid under conditions which remove the thiol-protectinggroup (see, e.g., U.S. Pat. No. 5,578,718). Thus, the nucleobase may bederivatized to include a thiol group, thereby allowing interaction withthe thiol-reactive group of a functionalized, encapsulated fluorescentnanocrystal, in forming thioether bonds that couple the nucleobase tothe functionalized, encapsulated fluorescent nanocrystal (e.g., anucleobase labeled with a functionalized, encapsulated fluorescentnanocrystal).

3.2(c) As yet another alternative, a relatively small (with respect tomol concentration of one or more other lipids used in the lipid mixture;e.g., 20:10:1; PC/ch/DPET) amount of a thiol group-containing lipid maybe present in the lipid mixture in producing the functionalized,encapsulated fluorescent nanocrystals. Such a lipid comprisesdistearolylphosphatidyl ethanolamidomethyl thioacetate (“DPET”). Thesurface groups of the lipid portion may be modified with hydroxylaminein deprotecting the thiol groups, thereby resulting in free thiolgroups. A cross-linking agent may be used which has a thiol-reactivegroup at one end and an amino-reactive group at the other end (see,e.g., 3.2 (b) herein). Thus, the functionalized, encapsulatedfluorescent nanocrystals having free thiol groups, a cross-linkingreagent, and a nucleobase having a free amino group are mixed togetherunder suitable conditions for the nucleobase to be labeled by thefunctionalized, encapsulated fluorescent nanocrystals. Nucleobases canbe derivatized with amino groups using methods known to those skilled inthe art. For example, the OH group located in the 3′ and/or 2′ positionof a nucleobase may be derivatized to include an amino group.Alternatively, a proparglyethoxyamino nucleoside may be used as achain-terminating nucleobase, wherein the reactive functionality of thischain-terminating nucleobase comprises the primary amino moiety or thesecondary amino moiety. As will be apparent to one skilled in the artfrom the descriptions herein, other combinations of reactivefunctionalities may be used to label an affinity molecule with afunctionalized, encapsulated fluorescent nanocrystal. For example, afunctionalized, encapsulated fluorescent nanocrystal comprising a thiolgroup-containing lipid (as described herein in 3.2 (c)) may be coupledto a thiol-derivatized affinity molecule comprising a nucleobase (asdescribed herein in 3.2 (b)) using a cross-linking reagent having athiol-reactive group at either end (e.g., 1,4-bis-maleimidobutane;1,4-bis-maleimidyl-2,3-dihydroxybutane).

EXAMPLE 4

This example further illustrates functionalized, encapsulatedfluorescent nanocrystals according to the present invention. Asdescribed in Example 3 herein in more detail, encapsulated fluorescentnanocrystals may be functionalized by comprising surface groupscomprising one or more reactive functionalities which are capable ofbonding to a reactive functionality of, and which may be used to bondthe liposome portion to, an affinity molecule. Thus, a combination ofreactive functionalities may be used to label an affinity molecule witha functionalized, encapsulated fluorescent nanocrystal. In Example 3, anaffinity molecule was illustrated as comprising a nucleobase. However,as apparent to one skilled in the art from the descriptions herein;other types of affinity molecules can similarly be coupled tofunctionalized, encapsulated fluorescent nanocrystals according to thepresent invention.

For example, and with the teachings of Example 3 in mind, the affinitymolecule may comprise a protein (e.g., a glycoprotein, peptide,lipoprotein, monoclonal antibody, an antibody fragment with bindingspecificity, a lectin, avidin, and the like) having a reactivefunctionality comprising an amine group which can be used to couple tothe reactive functionality of functionalized, encapsulated fluorescentnanocrystals using methods known in the art. Alternatively, the proteinmay comprise a reactive functionality comprising one or more free thiolgroups. To continue with this illustration, sulfhydryl (thiol) groups ofan antibody may be produced by reduction with a thiol reagent. Forexample, the antibody (e.g., 20 mg/ml in buffer, pH 8.7) may be treatedwith 2-mercaptoethanol (e.g., final concentration of 25 mM) undersuitable conditions (e.g., 4° C. for 10 minutes) to reduce the proteinto contain free thiol groups. The treated antibody may be purified(e.g., by size exclusion), and the number of sulfhydryl groups per moleof antibody can be determined (e.g., Ellman reaction); and thus,preferably an antibody having 2 to 3 free thiol groups is then reactedin a coupling reaction with the functionalized, encapsulated fluorescentnanocrystals (e.g., with or without a coupling reagent, depending on thereactive functionalities and the coupling reaction used). Other reducingagents, such as dithiothreitol, may be used to reduce a disulfide groupof an antibody or Fab fragment to a reactive functionality comprising afree sulfhydryl group. As yet another alternative, the protein maycomprise a reactive functionality comprising one or more free carboxylgroups. In one illustration, and using methodology described in moredetail in Example 3 herein, a free carboxyl group of the protein may beesterified which then allows coupling, in a coupling reaction, with afree amino group of the functionalized, encapsulated fluorescentnanocrystals.

In another preferred embodiment, the affinity molecule which is labeledwith functionalized, encapsulated fluorescent nanocrystals comprises anucleic acid molecule (e.g., oligonucleotide, primer, probe, aptamer,vector, molecular probe, and the like). For example, by adjustingfactors which include, but are not limited to, the number of freereactive functionalities per molecule, the ratio and/or number ofdifferent molecules to be coupled per coupling reaction, and acombination thereof, a functionalized, encapsulated fluorescentnanocrystal may be produced which is selected from the group consistingof a nucleic acid molecule labeled with a plurality of functionalized,encapsulated fluorescent nanocrystals, a nucleic acid molecule labeledwith a single functionalized, encapsulated fluorescent nanocrystal, afunctionalized, encapsulated fluorescent nanocrystal labeled with aplurality of nucleic acid molecules, and a combination thereof. As anillustrative example, the nucleic acid molecule may comprise a reactivefunctionality comprising one or more free amine groups. For example,using methodology described in more detail in Example 3 herein, reactivefunctionalities of nucleic acid molecules comprising free amino groups,and functionalized, encapsulated fluorescent nanocrystals having surfacegroups comprising free thiol groups are mixed together in the presenceof a component for substitution comprising a cross-linking reagent. Thecross-linking reagent has an amino-reactive group at one end, and athiol-reactive group at the other end. In an alternative embodiment,nucleic acid molecules comprising reactive functionalities comprisingfree amino-reactive groups (e.g., carboxyl groups) can be coupled tosurface groups comprising free amino groups of functionalized,encapsulated fluorescent nanocrystals using methods known in the art. Inyet another embodiment, the nucleic acid molecules comprise reactivefunctionalities comprising free thiol groups which can be coupled tosurface groups comprising a reactive functionality of functionalized,encapsulated fluorescent nanocrystals using methods known in the art(e.g., to thiol-reactive groups; or to free amino groups using across-linking reagent).

EXAMPLE 5

This example illustrates various embodiments for a method of strandsynthesis using functionalized, encapsulated fluorescent nanocrystalswhich comprise labeled nucleobases according to the present invention.Embodiments, other than those that are described herein for purposes ofillustration, for using the functionalized, encapsulated fluorescentnanocrystals according to the present invention in a method of strandsynthesis will be apparent to those skilled in the art from thedescriptions herein. In one embodiment, provided is a single setcomprised of at least four species of functionalized, encapsulatedfluorescent nanocrystals which are (a) efficiently excited by a singlelight source; (b) have closely spaced emission spectra that arespectrally resolvable (distinguishable) by peak emission wavelengths(e.g., allowing simultaneous detection of each individual peak); (c)have emissions of relatively high quantum efficiency; (d) are smallenough in size so as to minimize possible steric hinderance as relatedto incorporation and/or the progression during strand synthesis. Forexample, in one method of the strand synthesis comprising a modifiedSanger-type DNA sequencing protocol, utilized are at least four speciesof functionalized, encapsulated fluorescent nanocrystals having discretefluorescence emission spectra (e.g., one species is capable offluorescing red, one species is capable of fluorescing green, onespecies is capable of fluorescing yellow, one species is capable offluorescing orange) and comprising a different chain-terminatingnucleobase (e.g., one species comprises ddATP; one species comprisesddTTP, one species comprises ddCTP, one species comprises ddGTP) whichmay be incorporated into a synthesized strand. The species of labeledchain-terminating nucleobases are used in one or more sequencingreactions, followed by resolving the resultant differentially-labeledsynthesized strands (e.g., such as by size, length, or time), excitingthe synthesized strands with an excitation light source, and thenscanning for detection by a fluorimeter or other suitable detectionmeans that is capable of spectrally resolving the discrete fluorescencespectra of the excited functionalized, encapsulated fluorescentnanocrystals. Hence, the individual chain-terminating nucleobases may beidentified, and the sequence of the synthesized strand may bedetermined.

In another embodiment, the strand synthesis comprises employingfunctionalized, encapsulated fluorescent nanocrystals are utilized intoa sequencing protocol which relies on primer extension followed bybase-specific cleavage of primer extension products. In one example ofthis embodiment, a set of four different species of functionalized,encapsulated fluorescent nanocrystals (e.g., one species is capable offluorescing red, one species is capable of fluorescing green, onespecies is capable of fluorescing yellow, one species is capable offluorescing orange) and comprising a different nucleobase (e.g., onespecies comprises DATP; one species comprises dTTP, one speciescomprises dCTP, one species comprises dGTP) are incorporated duringstrand synthesis, and the synthesized strand is suspended in a movingfluid flow stream; an exonuclease is used to sequentially cleave anindividual nucleobase (labeled with a functionalized, encapsulatedfluorescent nanocrystal) from the end of the suspended synthesizedstrand, and each cleaved, labeled nucleobase is maintained in order ofcleavage for subsequent detection, spectral resolution, andidentification using an appropriate detection system in determining thesequence of a synthesized strand. As will be apparent to one skilled inthe art, another variation of this embodiment involves coupling afunctionalized, encapsulated fluorescent nanocrystal to the nucleobaseafter it is cleaved (post-replication, post-cleavage). A preferreddetection means may comprise a scanner or reader or other analyticalinstrument which can detect discrete fluorescence peaks that fall in aspectral range of from about 400 nm to about 900 nm; and, optionally(when more than one color is used in the detection system), distinguishbetween spectrally resolvable fluorescence peaks within that range.

In another embodiment, the functionalized, encapsulated fluorescentnanocrystals comprising labeled nucleobases are incorporated into a(nucleic acid) strand synthesized in a template-directed manner. Atemplate-directed manner is generally achieved by enzymatic copyingtemplate, and insertion of nucleobases in the strand synthesized, by anenzyme that replicates nucleic acids in a template-directed manner usingmethods known in the art. The strand synthesis may be a process selectedfrom the group consisting of nucleic acid amplification, fill-inreactions, reverse transcription, in vitro mutagenesis, cycled chaintermination sequence reactions, cycled primer extension reactions,random primer extension reactions, nick translations, primer elongation,methods for determining the presence and quantifying the number of di-and trinucleotide repeats, and DNA typing with short tandem repeatpolymorphisms.

EXAMPLE 6

This example illustrates various embodiments for using functionalized,encapsulated fluorescent nanocrystals which comprise labeled affinitymolecules according to the present invention. Embodiments, other thanthose that are described herein for purposes of illustration, for usingthe functionalized, encapsulated fluorescent nanocrystals according tothe present invention in a method of fluorescence detection will beapparent to those skilled in the art from the descriptions herein. In amethod of detection of a target substrate using the functionalizedencapsulated fluorescent nanocrystals according to the presentinvention, the functionalized encapsulated fluorescent nanocrystals areplaced in contact with a sample being analyzed for the presence orabsence of a substrate (“target substrate”) for which the affinitymolecule of the functionalized encapsulated fluorescent nanocrystals hasbinding specificity. Where the affinity molecule portion of thefunctionalized, encapsulated fluorescent nanocrystals comprises anucleic acid molecule, the method of detection of a target substratecomprises hybridization (when the target substrate is a nucleic acidmolecule) or binding (where the target substrate is a protein). Withrespect to hybridization, it is known in the art to refer to a processby which a single-stranded nucleic acid molecule joins with acomplementary strand through nucleotide base pairing. A sufficientnumber of complementary base pairs are needed for hybridization, and theselectivity of hybridization depends on the degree of complementarity,the stringency of conditions during the hybridization process, and thelength of the hybridizing strands. Thus, in a suitable detection system,a molecular probe comprising functionalized, encapsulated fluorescentnanocrystals is added in a diagnostically-effective amount to a samplebeing analyzed for the presence or absence of a target substrate insuitable conditions for the molecular probe to contact and bind totarget molecule if present in the sample. Typically, a wash step may beperformed to remove from the detection system any unbound ornonspecifically bound molecular probe, and then the sample is exposed toan appropriate excitation light source (e.g., depending on the nature ofthe fluorescent nanocrystals used), and detected (and may includequantitation) is any resultant fluorescence emission spectra.Quantitation of the amount of target substrate present is directlyrelated to the intensity of the emitted fluorescence peak. As known tothose skilled in the art of fluorescent nanocrystals, the absorbancepeak and fluorescence peak emissions depend on such factors whichinclude, but are not limited to, the chemical nature, the size ofsemiconductor nanocrystals, and amount of dopant comprising dMOnanocrystals. There are various assay system formats in which suchfunctionalized, encapsulated fluorescent nanocrystals may be used whichinclude, but are not limited to, Northern blot, Southern blot,microarrays (e. g., gene chips, protein chips), in-situ hybridization(“FISH”), screening of nucleic acid molecule libraries, geneticintroduction (e. g., transfection, infec-tion, electroporation)efficiency assays, molecular amplifi-cation assays, and assays for geneexpression.

In a preferred embodiment wherein a function-alized, encapsulatedfluorescent nanocrystal comprises one or more nucleic acid molecules,the nucleic acid molecule comprises an expression vector for expressionfrom a desired nucleic acid sequence, or a nucleic acid molecule (e.g.,gene), which is desired to be introduced (e.g., infection, transfection,electroporation) into a living cell. For example, the lipid mixturewhich is used to form the functionalized, encapsulated fluorescentnanocrystals comprises one or more cationic lipids, and one or more“helper lipids” in forming a lipid mixture which confers or facilitatestransfection efficiency of the resultant functionalized, encapsulatedfluorescent nanocrystals. Helper lipids are known to those skilled inthe art to include, but are not limited to,

dioleoylphosphatidylethanolamine (DOPE), choles-terol (ch),monooleoylglycerol, dioleoylphosphatidylcholine (DOPC), and acombination thereof. Cationic lipids are known to those skilled in theart to include, but are not limited to, 3 (beta) (N-(N&APOS;,N′-dimethylaminoethane)carba-moyl) cholesterol (DC-ch), N-(L-(2,3-dioleoyloxy)propyl-N, N, N-trimethylammonium chloride (DOTMA),dymyristyloxy-propyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), 1,2dioleoyl-3-trimethylammonium propane chloride (DOTAP), and a combinationthereof. It will be apparent to one skilled in the art that the lipidmixture may comprise one or more cationic lipids, or one or morecationic lipids in combination with one or more helper lipids, dependingon the desired performance and desired properties. For example, the oneor more cationic lipids may comprise from about 10% to about 90% or moreof the lipid mixture. Preferred lipid mixtures comprise: DMRIE and DOPE;MMCE and DOPE (1:1); DOTMA and ch (1:1); DOTAP and DC-ch; DMRIE andDC-ch; or DOPE and DC-ch. As apparent to one skilled in the art, thetransfection efficiency of a functionalized, encapsulated fluorescentnanocrystal may depend on one or more factors which include, but are notlimited to, the structure of the cationic lipids used in its formation,cationic lipid-to-nucleic acid molecule ratio, size of thefunctionalized, encapsulated fluorescent nanocrystal, and lipid mixturecontent.

In one embodiment, the functionalized, encapsulated fluorescentnanocrystals are mixed with nucleic acid molecule (including, but notlimited to, a plasmid, or other DNA molecule) in suitable ratios andamounts in forming complexes for promoting introduction into the cellsdesired to be transfected. For example, with respect to cell typesand/or transfection conditions in which a negatively charged complex isdesired, a relatively higher ratio of nucleic acid to functionalized,encapsulated fluorescent nanocrystals may be utilized. Likewise, forcell types and/or transfection conditions in which a positively chargedcompex is desired, a relatively higher ratio of functionalized,encapsulated fluorescent nanocrystals to nucleic acid molecule may beutilized. For example, depending on the cell type, the different ratiosof nucleic acid molecule:functionalized, encapsulated fluorescentnanocrystals may be used to form the complexes for transfection.Generally, these ratios are expressed in micrograms of nucleic acidmolecule:nanomoles of cationic lipid. For purposes of illustration, itis generally known that the ratio may range from about 15:1 to about1:20 (nucleic acid molecule:cationic lipid). Thus, for example, thenucleic acid molecule and the functionalized, encapsulated fluorescentnanocrystals are added together to form a mixture (e.g., by mixing, andincubating for 15 minutes at room temperature). Alternatively, thenucleic acid molecule may be encapsulated in the functionalized,encapsulated fluorescent nanocrystals by incorporating the nucleic acidmolecule (e.g., at the appropriate ratio) during liposome formation,including, but not limited to, in the dried lipid mixture film, or thesolution for hydrating the dried lipid mixture film. In eitherembodiment, the resultant complexes may then utilized in transfectionprotocols known in the art (e.g., may be added to media and the media isincubated with cells under suitable conditions for transfection, such as5–6 hours in a tissue culture incubator at 37° C. with CO₂). Followingthis process, the efficiency of introducing the functionalized,encapsulated fluorescent nanocrystals into the cells may be evaluated bysubjecting the cells to fluorescence analysis by a method which mayinclude, but is not limited to, fluorescence microscopy, or fluorescencescanner. For example, the cells are exposed to an appropriate excitationlight source (e.g., depending on the nature of the fluorescentnanocrystals used), and detected is any resultant fluorescence emissionspectra emitted from functionalized, encapsulated fluorescentnanocrystals. Quantitation of the amount of functionalized, encapsulatedfluorescent nanocrystals present in the cells is directly related to theintensity of the emitted fluorescence peak.

EXAMPLE 7

This example illustrates various embodiments for a method of usingfunctionalized, encapsulated fluorescent nanocrystals which compriselabeled affinity molecules according to the present invention.Embodiments, other than those that are described herein for purposes ofillustration, for using the functionalized, encapsulated fluorescentnanocrystals according to the present invention in a method offluorescence detection will be apparent to those skilled in the art fromthe descriptions herein. In a method of detection of a target substrateusing the functionalized encapsulated fluorescent nanocrystals accordingto the present invention, the functionalized encapsulated fluorescentnanocrystals are placed in contact with a sample being analyzed for thepresence or absence of a target substrate for which the affinitymolecule of the functionalized encapsulated fluorescent nanocrystals hasbinding specificity. In this embodiment, the affinity molecule portionof the functionalized, encapsulated fluorescent nanocrystals comprises aprotein with binding specificity (e.g., monoclonal antibody, peptide,lectin) or an aptamer with binding specificity. The functionalized,encapsulated fluorescent nanocrystals are contacted with the samplebeing analyzed for the presence or absence of a target substrate.Subsequent binding, between the affinity molecule portion of thefunctionalized, encapsulated fluorescent nanocrystals and the targetsubstrate, if present in the sample, in a detection system results incomplexes comprising the functionalized, encapsulated fluorescentnanocrystal-target sub-strate which can emit a detectable signal forquantitation, visualization, or other form of detection. Upon formationof the complexes comprising the functionalized, encapsulated fluorescentnanocrystal-substrate, the detectable signal emitted therefrom may bedetected by first exposing the complexes formed in the detection systemto an excitation spectra of light (UV or other suitable light source;depending on the nature of the fluorescent nanocrystal used) that issuitable for exciting the functionalized, encapsula-ted fluorescentnanocrystals to emit a fluorescence peak.

The peak is then detected, or detected and quantitated, by appropriatedetection means (e. g., photodetector, filters, fluorescence microscope,and the like). Quantitation of the amount of target substrate present isdirectly related to the intensity of the emitted fluorescence peak. Asknown to those skilled in the art of fluorescent nanocrystals, theabsorbance peak and fluorescence peak emissions depend on such factorswhich include, but are not limited to, the chemical nature, and amount,of the dopant comprising dMO nanocrystals; and the chemical nature andsize of semicon-ductor nanocrystals. As will be apparent to one skilledin the art, the detection system may include, but is not limited to, afluorescence-based immunoassay, fluorescence-based detection systems,fluorescent staining (e.g., immuno-fluorescent staining on a glassslide), microarrays, flow cytometry, molecular tracking (e.g., locatingor tracking a target substrate), molecular sorting (e.g., cell sortingby flow cytometry), fluorescence imaging (e.g., of live tissue, or fiberoptic fluorescence imaging microscopy), and the like.

EXAMPLE 8

In another illustration of a method for using functionalized,encapsulated fluorescent nanocrystals, it may be desirable to quench(reduce or eliminate) the fluorescence signal emitted by functionalized,encapsulated fluorescent nanocrystals present in a detection system(particularly a detection system which may use an aqueous solution;e.g., an immunoassay, microarray, and the like). To illustrate thisexample, and wherein the functionalized, fluorescent nanocrystals arepresent in an aqueous-based solution (either before or after treatmentwith the lipolytic agent), the liposome portion of the functionalized,encapsulated fluorescent nanocrystals may be disrupted to release thefluorescent nanocrystals. The fluorescent nanocrystals, being insolublein aqueous solutions, can form aggregates when they interact togetherwhich can cause irreversible flocculation of the fluorescentnanocrystals. Thus, a method of quenching fluorescence, in a detectionsystem, from a substrate comprising functionalized, encapsulatedfluorescent nanocrystals comprises: contacting the functionalized,encapsulated fluorescent nanocrystals with a lipolytic agent in aneffective amount to disrupt liposome portions of the functionalized,encapsulated fluorescent nanocrystals in releasing fluorescentnanocrystals; and removing the released fluorescent nanocrystals fromthe detection system (e.g., by allowing the released fluorescentnanocrystals to precipitate in an aqueous-based solution therebyremoving it from the substrate, and/or washing the released fluorescentnanocrystals from the detection system) in quenching the fluorescence.

Also, when the liposome portion of the functionalized, encapsulatedfluorescent nanocrystals is disrupted, the released fluorescentnanocrystals may be washed from the system (using a solution appropriatefor the detection system, as is standard in the art). For example, wherethe affinity molecule portion of the functionalized, encapsulatedfluorescent nanocrystals is bound to a target substrate in the system,thereby immobilizing the functionalized, encapsulated fluorescentnanocrystals, disruption of the liposome portion will release thefluorescent nanocrystals from being immobilized. As apparent to oneskilled in the art, there are several means by which the liposomeportion of functionalized, encapsulated fluorescent nanocrystals may bedisrupted. For example, a lipolytic agent for disrupting the liposomeportion includes, but is not limited to, a lipolytic enzyme, componentsof the complement system sufficient to cause complement-mediated lysis,a detergent (e.g., a non-ionic detergent such as TRITON X-100; saponin;and the like), a water-miscible alcohol, and a combination thereof.Briefly, the functionalized, encapsulated fluorescent nanocrystals arecontacted with an amount of the lipolytic agent effective to causedisruption (breakage or lysis) of the functionalized, encapsulatedfluorescent nanocrystals. Depending on the lipolytic agent, and the timein which to achieve the desired disruption, an amount of a lipolyticagent effective to cause such lysis or disruption may comprise fromabout 10% to about 90% of the volume of the aqueous-based solutioncomprising the functionalized, encapsulated fluorescent nanocrystals tobe disrupted. The released fluorescent nanocrystals may then be removedor excluded from the detection assay or system.

EXAMPLE 9

As previously described herein in more detail, a method of makingfunctionalized, encapsulated fluorescent nanocrystals according to thepresent invention comprises the steps of: (a) mixing fluorescentnanocrystals with a lipid mixture comprising the lipids (e.g., one ormore phospholipids, or one or more phospholipids and one or moresterols) to form a dried lipid mixture film; (b) contacting the driedlipid mixture film with an aqueous solution; and (c) mixing the driedlipid mixture film with the aqueous solution in forming functionalized,encapsulated fluorescent nanocrystals. The mixing step (c) may beperformed by any method known in the art which includes, but is notlimited to, vortexing, sonication, applying pressure, extrusion,injection, and a combination thereof. As apparent from the descriptionsherein, the dried lipid mixture film comprises the lipids desired toform the liposome portion of the functionalized, encapsulatedfluorescent nanocrystals; and in an alternative embodiment, the lipidmixture may further comprise one or more components for substitution. Asapparent from the descriptions herein, the aqueous solution may furthercomprise one or more components for substitution, affinity molecule, ora combination thereof. The method according to the present invention mayfurther comprise post-treating the functionalized, encapsulatedfluorescent nanocrystals by contacting the functionalized, encapsulatedfluorescent nanocrystals with one or more components for substitutionunder suitable conditions for the one or more components forsubstitution to become part of the liposome portion of thefunctionalized, encapsulated fluorescent nanocrystals. As apparent fromthe descriptions herein, the one or more components for substitutioncomprises one or more affinity molecules which have a reactivefunctionality that is coupled to the reactive functionality of theliposome portion of the functionalized, encapsulated fluorescentnanocrystals. Additionally, the method may further comprise apurification step (e.g., size exclusion, density separation, separationbased on solubility, magnetic separation based on magnetic attraction ofthe encapuslated fluorescent nanocrystals to a magnet source, and thelike) to purify the desired population of functionalized, encapsulatedfluorescent nanocrystals. As previously described herein in more detail,one embodiment of mixing fluorescent nanocrystals with a lipid mixtureto form a dried lipid mixture film comprises mixing the fluorescentnanocrystals and lipid mixture in the same organic solvent, and thenevaporating the solvent to form the dried lipid mixture film.Alternatively, the fluorescent nanocrystals in a solvent may beevaporated to form a dried preparation of fluorescent nanocrystals, thelipid mixture may be dried, and then the dried preparation offluorescent nanocrystals and dried preparation of lipid mixture may bemixed together to form the dried lipid mixture film.

The foregoing description of the specific embodiments of the presentinvention have been described in detail for purposes of illustration. Inview of the descriptions and illustrations, others skilled in the artcan, by applying, current knowledge, readily modify and/or adapt thepresent invention for various applications without departing from thebasic concept, and therefore such modifications and/or adaptations areintended to be within the meaning and scope of the appended claims.

1. A functionalized, encapsulated fluorescent nanocrystal comprising (a)liposome; (b) one or more semiconductor fluorescent nanocrystalsencapsulated by the liposome; and (c) surface groups, wherein an outersurface of the liposome comprises the surface groups, and wherein thesurface groups are selected from the group consisting of one or morereactive functionalities, one or more affinity molecules, and acombination thereof.
 2. A functionalized, encapsulated fluorescentnanocrystal comprising (a) a liposome; (b) one or more doped metal-oxidefluorescent nanocrystal encapsulated by the liposome; and (c) surfacegroups, wherein an outer surface of the liposome comprises the surfacegroups, and wherein the surface groups are selected from the groupconsisting of one or more reactive functionalities, one or more affinitymolecules, and a combination thereof.
 3. A functionalized, encapsulatedfluorescent nanocrystal comprising (a) a liposome; (b) a plurality offluorescent nanocrystals comprising one or more doped metal-oxidenanocrystals and one or more semiconductor nanocrystals, the fluorescentnanocrystal encapsulated by the liposome; and (c) surface groups,wherein an outer surface of the liposome comprises the surface groups,and wherein the surface groups are selected from the group consisting ofone or more reactive functionalities, one or more affinity molecules,and a combination thereof.
 4. The functionalized, encapsulatedfluorescent nanocrystal according to claim 1, wherein the liposomefurther comprises a component for substitution selected from the groupconsisting of a membrane stabilizer, an isotonic agent, a pH adjustingagent, an aggregation minimizer, an affinity molecule, an amino acid,and a combination thereof.
 5. The functionalized, encapsulatedfluorescent nanocrystal according to claim 4, wherein the liposomecomprises a membrane stabilizer selected from the group consisting ofone or more sterols, one or more fatty acids, one or more amino acids,and a combination thereof.
 6. The functionalized, encapsulatedfluorescent nanocrystal according to claim 5, wherein the liposomecomprises one or more phospholipids and one or more sterols.
 7. Thefunctionalized, encapsulated fluorescent nanocrystal according to claim3, wherein the surface groups comprise a reactive functionalitycomprising free amino groups.
 8. The functionalized, encapsulatedfluorescent nanocrystal according to claim 3, wherein the surface groupscomprise a reactive functionality comprising thiol-reactive groups. 9.The functionalized, encapsulated fluorescent nanocrystal according toclaim 1, wherein the surface groups comprise a reactive functionalitycomprising free thiol groups.
 10. The functionalized, encapsulatedfluorescent nanocrystal according to claim 3, wherein the surface groupscomprise a reactive functionality comprising free carboxyl groups. 11.The functionalized, encapsulated fluorescent nanocrystal according toclaim 1, wherein the liposome is comprised of one or more cationiclipids and one or more helper lipids in forming a liposome adapted fortransfection and one or more nucleic acid molecules.
 12. Thefunctionalized, encapsulated fluorescent nanocrystal according to claim3, wherein the liposome is comprised of one or more cationic lipids informing a liposome adapted for transfection and one or more nucleic acidmolecules.
 13. A method of using functionalized, encapsulatedfluorescent nanocrystals according to claim 1 in a detection system,wherein the surface groups comprise an affinity molecule, the methodcomprising the steps of: (a) contacting the functionalized, encapsulatedfluorescent nanocrystals with a sample being analyzed for the presenceor absence of a substrate for which the affinity molecule has bindingspecificity, wherein if the substrate is present in the sample, formedare complexes comprising the functionalized, encapsulated fluorescentnanocrystals bound to the substrate; (b) exposing the complexes, ifformed, in the detection system to an excitation light source suitablefor exciting the functionalized encapsulated fluorescent nanocrystals toemit a fluorescence peak; and (c) detecting the fluorescence peakemitted by the complexes, if present, by a detection means for detectingthe fluorescence peak; wherein the detection of a fluorescence peak isindicative of the presence of the substrate.
 14. The method according toclaim 13, wherein the presence of the substrate is detected, and furthercomprises quantitating the amount of substrate present by measuring theintensity of the fluorescence peak emitted.
 15. The method according toclaim 13, wherein the affinity molecule comprises a nucleic acidmolecule, and wherein the detection system comprises hybridization. 16.The method according to claim 13, wherein the detection system isselected from the group consisting of a fluorescence-based immunoassay,fluorescence-based detection systems, microarrays, fluorescent staining,flow cytometry, strand synthesis, molecular sorting, molecular tracking,and fluorescence imaging.
 17. The method according to claim 13, whereinthe affinity molecule comprises a monoclonal antibody.
 18. The methodaccording to claim 13, wherein the surface groups comprise a nucleobase,and wherein the detection system comprises strand synthesis in whichfunctionalized encapsulated fluorescent nanocrystals are incorporatedinto a nucleic acid strand synthesized in a template-directed manner.19. The method according to claim 13, wherein fluorescence is detected,the method further comprising contacting the functionalized,encapsulated fluorescent nanocrystals in the detection system with alipolytic agent in an effective amount to disrupt liposome portions ofthe functionalized, encapsulated fluorescent nanocrystals in releasingfluorescent nanocrystals; and removing the released fluorescentnanocrystals from the detection system so as to quench the fluorescence.20. A method of making functionalized encapsulated fluorescentnanocrystals, the method comprising: (a) mixing fluorescent nanocrystalswith a lipid mixture to form a dried lipid mixture film; (b) contactingthe dried lipid mixture film with an aqueous solution; and (c) mixingthe dried lipid mixture film with the aqueous solution in formingfunctionalized, encapsulated fluorescent nanocrystals.
 21. The methodaccording to claim 20, wherein the fluorescent nanocrystals and lipidmixture are mixed in an organic solvent, and the organic solvent isevaporated to form the dried lipid mixture.
 22. The method according toclaim 20, wherein an organic solvent containing fluorescent nanocrystalsis evaporated in forming a dried preparation of fluorescentnanocrystals, and an organic solvent containing the lipid mixture isevaporated in forming a dried preparation of lipid mixture; and thedried preparation of fluorescent nanocrystals and the dried preparationof lipid mixture are mixed to form the dried lipid mixture.
 23. Themethod according to claim 20, wherein the fluorescent nanocrystalscomprise semiconductor nanocrystals.
 24. The method according to claim20, wherein the fluorescent nanocrystals comprise doped metal oxidenanocrystals.
 25. The method according to claim 20, wherein thefluorescent nanocrystals comprise semiconductor nanocrystals and dopedmetal oxide nanocrystals.
 26. The method according to claim 20, whereinthe dried lipid mixture film further comprises a component forsubstitution selected from the group consisting of a membranestabilizer, an isotonic agent, a pH adjusting agent, an aggregationminimizer, an affinity molecule, an amino acid, and a combinationthereof.
 27. The method according to claim 20, wherein the aqueoussolution further comprises a component for substitution selected fromthe group consisting of a membrane stabilizer, an isotonic agent, a pHadjusting agent, an aggregation minimizer, an affinity molecule, anamino acid, and a combination thereof.
 28. The functionalizedencapsulated fluorescent nanocrystals according to claim 2, wherein theliposome further comprises a component for substitution selected fromthe group consisting of a membrane stablizer an isotonic agent a pHadjusting agent, an aggregation minimizer, an affinity molecule, anamino acid, and a combination thereof.
 29. The functionalizedencapsulated fluorescent nanocrystals according to claim 28, wherein theliposome further comprises a component for substitution selected fromthe group consisting of a membrane stabilizer, an isotonic agent, a pHadjusting agent, an aggregation minimizer, an affinity molecule, anamino acid, and a combination thereof.
 30. The functionalizedencapsulated fluorescent nanocrystals according to claim 29, wherein theliposome comprises a membrane stabilizer selected from the groupconsisting of one or more sterols, one or more fatty acids, one or moreamino acids, and a combination thereof.
 31. The functionalizedencapsulated fluorescent nanocrystals according to claim 30, wherein theliposome comprises one or more phospholipids and one or more sterols.32. The functionalized encapsulated fluorescent nanocrystals accordingto claim 28, wherein the surface group comprise a reactive functionalitycomprising free amino groups.
 33. The functionalized encapsulatedfluorescent nanocrystals according to claim 28, wherein the surfacegroups comprise a reactive functionality comprising thiol reactivegroups.
 34. The functionalized encapsulated fluorescent nanocrystalsaccording to claim 28, wherein the surface groups comprise a reactivefunctionality comprising free thiol groups.
 35. The functionalizedencapsulated fluorescent nanocrystals according to claim 28, wherein thesurface groups comprise a reactive functionality comprising freecarboxyl groups.
 36. The functionalized encapsulated fluorescentnanocrystals according to claim 28, wherein the liposome is comprised ofone or more cationic lipids and one or more helper lipids in forming aliposome adapted for transfection and one or more nucleic acidmolecules.
 37. The functionalized encapsulated fluorescent nanocrystalsaccording to claim 28, wherein the liposome is comprised of one or morecationic lipids in forming a liposome adapted for transfection and oneor more nucleic acid molecules.
 38. A method of using functionalizedencapsulated fluorescent nanocrystals according to claim 28, in adetection system, wherein the surface groups comprise an affinitymolecule, the method comprising the steps of: (a) contacting thefunctionalized encapsulated fluorescent nanocrystals with a sample beinganalyzed for the presence or absence of a substrate for which theaffinity molecule has binding specificity, wherein if the substrate ispresent in the sample, formed are complexes comprising thefunctionalized encapsulated fluorescent nanocrystals bound to thesubstrate; (b) exposing the complexes if formed in the detection systemto an excitation light source suitable for exciting the functionalizedencapsulated fluorescent nanocrystals to emit a fluorescence peak; and(c) detecting the fluorescence peak emitted by the complexes, ifpresent, by a detection means for detecting the fluorescence peak;wherein the detection of a fluorescence peak is indicative of thepresence of the substrate.
 39. The method according to claim 38, whereinthe presence of the substrate is detected, and further comprisesquantitating the amount of substrate present by measuring the intensityof the fluorescence peak emitted.
 40. The method according to claim 38,wherein the affinity molecule comprises a nucleic acid molecule, andwherein the detection system comprises hybridization.
 41. The methodaccording to claim 38, wherein the detection system is selected from thegroup consisting of a fluorescence based immunoassay, fluorescence-baseddetection systems, microassays, fluorescent staining, flow cytometrystrand synthesis, molecular sorting, molecular tracking, andfluorescence imaging.
 42. The method according to claim 38, wherein theaffinity molecule comprises a monoclonal antibody.
 43. The methodaccording to claim 38, wherein the surface groups comprise a nucleobase,and wherein the detection system strand synthesis in whichfunctionalized encapsulated fluorescent nanocrystals are incorporatedinto a nucleic acid strand synthesized in a template-directed manner.44. The method according to claim 38, wherein fluorescence is detectedthe method further comprising contacting the functionalized encapsulatedfluorescent nanocrystals in the detection system with a lipolytic agentin an effective amount to disrupt liposome portions of thefunctionalized encapsulated fluorescent nanocrystals in releasingfluorescent nanocrystals; and removing the released fluorescentnanocrystals from the detection system so as to quench the fluorescence.45. The functionalized encapsulated fluorescent nanocrystals accordingto claim 3, wherein the liposome further comprises a component forsubstitution selected from the group consisting of a membranestabilizer, an isotonic agent, a pH adjusting agent, an aggregationminimizer, an affinity molecule, an amino acid, and a combinationthereof.
 46. The functionalized encapsulated fluorescent nanocrystalsaccording to claim 45, wherein the liposome further comprises acomponent for substitution selected from the group consisting of amembrane stabilizer, an isotonic agent, a pH adjusting agent, anaggregation minimizer, an affinity molecule, an amino acid, and acombination thereof.
 47. The functionalized encapsulated fluorescentnanocrystals according to claim 46, wherein the liposome comprises amembrane stabilizer, selected from the group consisting of one or moresterols, one or more fatty acids, one or more an amino acids, and acombination thereof.
 48. The functionalized encapsulated fluorescentnanocrystals according to claim 47, wherein the liposome comprises oneor more phospholipids and one or more sterols.
 49. The functionalizedencapsulated fluorescent nanocrystals according to claim 45, wherein thesurface groups comprise a reactive functionality comprising free aminogroups.
 50. The functionalized encapsulated fluorescent nanocrystalsaccording to claim 45, wherein the surface groups comprise a reactivefunctionality comprising thiol-reactive groups.
 51. The functionalizedencapsulated fluorescent nanocrystals according to claim 45, wherein thesurface groups comprise a reactive functionality comprising free thiolgroups.
 52. The functionalized encapsulated fluorescent nanocrystalsaccording to claim 45, wherein the surface groups comprise a reactivefunctionality comprising free carboxyl groups.
 53. The functionalizedencapsulated fluorescent nanocrystals according to claim 45, wherein theliposome is comprised of one or more cationic lipids and one or morehelper lipids in forming a liposome adapted for transfection and one oremore nucleic acid molecule.
 54. The functionalized encapsulatedfluorescent nanocrystals according to claim 45, wherein the liposome iscomprised of one or more cationic lipids in forming a liposome adaptedfor transfection and one or more nucleic acid molecules.
 55. A method ofusing functionalized, encapsulated fluorescent nanocrystals according toclaim 45, in a detection system, wherein the surface groups comprise anaffinity molecule the method comprising the steps of: (a) contacting thefunctionalized, encapsulated fluorescent nanocrystals with a samplebeing analyzed for the presence or absence of a substrate for which theaffinity molecule has binding specificity, wherein if the substrate ispresent in the sample, formed are complexes comprising thefunctionalized, encapsulated fluorescent nanocrystals bound to thesubstrate; (b) exposing the complexes, if formed in the detection systemto an excitation light source suitable for exciting the functionalizedencapsulated fluorescent nanocrystals to emit a fluorescence peak; and(c) detecting the fluorescence peak emitted by the complexes, if presentby a detection means for detecting the fluorescence peak, wherein thedetection of a fluorescence peak is indicative of the presence of thesubstrate.
 56. The method according to claim 55, wherein the presence ofthe substrate is detected, and further comprises quantitating the amountof substrate present by measuring the intensity of the fluorescence-peakemitted.
 57. The method according to claim 55, wherein the affinitymolecule comprises a nucleic acid molecule, and wherein the detectionsystem comprises hybridization.
 58. The method according to claim 55,wherein the detection system is selected from the group consisting of afluorescence-based immunoassay, fluorescence-based detection systems,microarrays, fluorescent-staining, flow cytometry, strand synthesis,molecular sorting, molecular tracking, and fluorescence imaging.
 59. Themethod according to claim 55, wherein the affinity molecule comprises amonoclonal antibody.
 60. The method according to claim 55, wherein thesurface groups comprise a nucleobase, and wherein the detection systemcomprises strand synthesis in which functionalized encapsulatedfluorescent nanocrystals are incorporated into a nucleic acid strandsynthesized in a template directed manner.
 61. The method according toclaim 55, wherein fluorescence is detected the method further comprisingcontacting the functionalized, encapsulated fluorescent nanocrystals inthe detection system with a lipolytic agent in an effective amount todisrupt liposome portions of the functionalized, encapsulatedfluorescent nanocrystals in releasing fluorescent nanocrystals andremoving the released fluorescent nanocrystal from the detection systemso as to quench the fluorescence.